Figures

Abstract

Background

Immunization of mice with the Trypanosoma cruzi trans-sialidase (TS) gene using plasmid DNA, adenoviral vector, and CpG-adjuvanted protein delivery has proven highly immunogenic and provides protection against acute lethal challenge. However, long-term protection induced by TS DNA vaccines has not been reported. The goal of the present work was to test whether the co-administration of a plasmid encoding IL-15 (pIL-15) could improve the duration of protection achieved through genetic vaccination with plasmid encoding TS (pTS) alone.

Methodology

We immunized BALB/c mice with pTS in the presence or absence of pIL-15 and studied immune responses [with TS-specific IFN-γ ELISPOT, serum IgG ELISAs, intracellular cytokine staining (IFN-γ, TNF-α, and IL-2), tetramer staining, and CFSE dilution assays] and protection against lethal systemic challenge at 1 to 6 months post vaccination. Mice receiving pTS alone developed robust TS-specific IFN-γ responses and survived a lethal challenge given within the first 3 months following immunization. The addition of pIL-15 to pTS vaccination did not significantly alter T cell responses or protection during this early post-vaccination period. However, mice vaccinated with both pTS and pIL-15 challenged 6 months post-vaccination were significantly more protected against lethal T. cruzi challenges than mice vaccinated with pTS alone (P<0.05). Improved protection correlated with significantly higher numbers of TS-specific IFN-γ producing total and CD8+ T cells detected>6 months post immunization. Also, these TS-specific T cells were better able to expand after in vitro re-stimulation.

Conclusion

Author Summary

Over 11 million individuals are infected with Trypanosoma cruzi, the causative agent of Chagas disease, which kills >50,000 people annually. Although recent vector control efforts and increased use and effectiveness of chemotherapeutic drugs including benznidazole have reduced infection rates and mortality, a safe, effective vaccine is needed. Vaccination with the T. cruzi trans-sialidase (TS) has been used effectively in mice to reduce mortality and chronic disease, however, the establishment of vaccine-induced long-term protective immunity remains elusive. Co-immunization strategies utilizing immune regulators such as interleukin-12 (IL-12) and interleukin-15 (IL-15) can be used to enhance antigen-specific T cell responses and prolong protective immunity. In the present report, we show that genetic vaccination of BALB/c mice with plasmid DNA encoding both TS and IL-15 compared with plasmid DNA encoding TS alone significantly enhanced CD4+ and CD8+ T cell responses including increased TNF-α, IFN-γ, and IL-2 production, and long-term protection against lethal systemic parasite challenge.

Funding: This work was supported by the National Institutes of Health grant RO1 AI040196 to DFH. MMR and OBR were supported by grants from Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, The National Institute for Vaccine Technology (INCTV-CNPq), The Millennium Institute for Vaccine Development and Technology (CNPq - 420067/2005-1), and The Millennium Institute for Gene Therapy. MMR and OBR are both recipients of fellowships from CNPq. JRV is a FAPESP fellowship recipient (Fundacao de Amparo a pesquisa do Estado de Sao Paulo). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Over 11 million individuals are infected worldwide with Trypanosoma cruzi, and over 50,000 deaths are attributed to this protozoan parasite annually [1]. Human infection usually occurs through contact with T. cruzi contaminated triatomine excreta, although the parasite also can be transmitted via congenital and parenteral routes or by organ donation [2]. Acute infection, usually associated with only mild and nonspecific symptoms including fever, fatigue, and high parasitemia, often goes undiagnosed. Although high level parasitemia and symptoms resolve after 3–8 weeks, low level tissue parasitism persists for decades, leading to pathologic manifestations of Chagas disease including cardiomyopathy and the mega-syndromes (mega-esophagus and mega-colon) in 30–50% of chronically infected individuals. Chemotherapy with benznidazole and nifurtimox can be highly successful if used within the first several weeks of infection, but rarely leads to cure during chronic infection, and toxic side effects limit their use. Because of the high risk of infection and disease in endemic countries and the lack of well tolerated trypanocidal drugs, a safe and effective vaccine is needed.

Several T. cruzi antigens, including trans-sialidase (TS), have been used successfully as experimental vaccines to reduce acute T. cruzi infection and chronic inflammation in mice [3]–[10]. TS, a member of the largest T. cruzi gene family (>1400 genes), is expressed on blood form trypomastigotes (BFT) and metacyclic trypomastigotes (MT), and is also present during early and late stage intracellular infection [11], [12]. T. cruzi is unable to synthesize sialic acid, which seems to be required for parasite infectivity. TS cleaves sialic acid from host cell donor molecules and transfers the released sialic acid onto the parasite surface. Vaccines incorporating TS (expressed in adenoviral vectors, in DNA vaccines, or purified recombinant protein mixed with toll-like receptor-9 CpG motifs) induce robust CD4+ and CD8+ T cell as well as antibody responses in mice, and more importantly, can protect against lethal T. cruzi challenge [3], [13]. However, only short term TS vaccine-induced protection has been reported.

Interleukin-15 (IL-15) is one of the so-called ‘homeostatic’ cytokines, which is beneficial for long-term survival of memory T cells. The functions of IL-15 are similar to that of IL-2, although these two cytokines do not have amino acid homology. IL-15 is synthesized by many types of cells including monocytes, macrophages, epithelial, and dendritic cells, but is not expressed by T cells. Central memory T cells (Tcm) express the receptor for IL-15 (IL-15R), and have the capacities to both extensively proliferate and produce key effector molecules after antigenic restimulation. These central memory T cells are critical for long-term protective immunity. In this work, we describe the enhancement of long term CD8+ T cell responses and protective T. cruzi immunity in mice (>6 months post vaccination) after vaccination with a combination of DNA vaccines encoding both TS and IL-15.

Materials and Methods

Ethics

All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC)/Animal Care Committee (ACC) at Saint Louis University. The University is registered with the USDA as a research facility (43-R-011), is regularly inspected and files an annual report. In addition, under the provisions of the Public Health Service Policy on the Humane Care and Use of Laboratory Animals, revised September 1986, the University has filed the appropriate assurance documents. (Assurance number A-3225-01). The facilities and programs for the use of animals at Saint Louis University are FULLY ACCREDITED by the American Association for the Accreditation of Laboratory Animal Care (AAALAC). The date of our most recent notification was June 18, 2009.

Parasites and mice

Six to eight week old female BALB/c mice were used throughout these studies (Charles River/NCI). Tulahuén strain T. cruzi blood form trypomastigotes (BFT) were prepared by bi-weekly passage through BALB/c mice as previously described [14], [15].

Optimized IL-15 gene construction

The murine interleukin-15 (IL-15) gene (genbank accession number BC023698) containing the long signal peptide (LSP) was cloned into the Invitrogen pVAX-1 vector (pIL-15LSP), and used as the PCR template for subsequent subcloning strategies. The LSP of IL-15 was replaced with an optimized signal peptide incorporating an efficient Kozak sequence and the 18aa signal peptide derived from Homo Sapiens immunoglobulin secretory leader signal as previously described [16]–[18]. This was accomplished using PCR primers 5′-GCCCCCGGATCCGCCGCCACCATGGATTGGACTTGGATCTTATTTTTAGTTGCTGCTGCTACTAGAGTTCATTCTAACTGGATAGATGTAAGATAT-3′ and 5′-GCCCCCGAATTCTCAGGACGTGTTGATGAACATTTG-3′. The resulting PCR product was subcloned into pVAX-1 using BamH1 and EcoR1 restriction sites (pIL-15opt, later referred to as pIL-15). Plasmids (pVAX, pIL-15LSP, and pIL-15opt) were propagated in DH5α strain E. coli and purified using QIAGEN plasmid purification kits (Valencia, CA).

Measurement of IL-15 expression in COS-7 cells

COS-7 cells (ATCC, Manassas, VA) were transfected with pVAX, pIL-15LSP, or pIL-15opt using Lipofectamine 2000 transfection reagent (Invitrogen) as recommended by the manufacturer. After 48 hours, cell lysates were prepared for use in IL-15-specific western blots and IL-15 bioassays. Western blots were performed using biotinylated goat anti-mouse IL-15 and streptavidin-HRP, followed by development with Luminol chemiluminescent substrate. In addition, lysates from transfected COS-7 cells were added to 96 well flat bottom plates containing 5×103 CTLL-2 cells (IL-15 sensitive cell line, ATCC) and incubated at 37°C for 24 hours prior to the addition of 0.5 µCi [3H] thymidine. After 24 hours, cells were harvested onto glass fiber filtermats using a Tomtec Mach-IIIM automated cell harvester, Ultima Gold F scintillation fluid added, and radioactive incorporation measured using a Wallac Microbeta 1450 scintillation counter [3]. A standard curve of mIL-15 was generated using purified rmIL-15, and employed to determine levels of functional IL-15 present in transfected COS-7 cell lysates (reported in ng/ml).

Spleen cells (2×106/ml) from mice vaccinated 6 months prior were stimulated with 1×105/ml A20J cells or A20J transfected with TS (A20-TS) in round bottom culture tubes. After overnight culture, monensin and brefeldin-A were added as recommended by the manufacturer (GolgiPlug and GolgiStop, respectively, BD Biosciences). In order to study polyclonal T cell responses, cells were rested overnight prior to addition of phorbol-12-myristate 13-acetate (PMA, 10 ng/ml), ionomycin (500 ng/ml), monensin, and brefeldin-A for the final 3 hours of culture. Cells were then washed with PBS, stained using LIVE/DEAD fixable Aqua Stain cell reagent, (Invitrogen), Fc receptors blocked by incubation with anti-CD16/32 (15 minutes, 4oC), then surface stained at 4oC for 30 minutes using CD3-FITC, CD8-PerCP, CD4-Pacific Blue, and TS(IYNVGQVSI)/H2Kd-tetramer-APC (all antibodies were purchased from BD Biosciences unless noted otherwise, and the IYNVGQVSI-specific APC tetramer reagent was provided through the NIH Tetramer Core Facility, Atlanta, GA). BD Cytoperm/Cytofix was used to fix and permeablize the cells as per manufacturer suggestions prior to staining with IL-2-PE, IFN-γ-Alexa700 and TNF-α-PE-Cy7 (30 minutes at 4oC). Cells were then washed and resuspended in permeabilization buffer prior to acquisition on a LSR-II flow cytometer (BD). Data sets were analyzed using FLOWJO Flow Cytometry Analysis v7 software (Tree Star, Inc., Ashland, OR).

Analysis of TS(IYNVGQVSI)/H2Kd tetramer positive cells

Six months following immunization, spleen cells were stained directly ex vivo using the surface staining procedure described above. Additionally, spleen cells were stained with CFSE (Vybrant CFDA SE cell tracer, Invitrogen), washed 3 times, and cultured (2×106/ml) with irradiated (12,500 rads) A20J or A20-TS cells (1×105) for 6 days at 37oC using the same media as described above (T-cell+). Cells were then washed and stained for CD3, CD8, CD4, and TS(IYNVGQVSI)/H2Kd tetramer binding prior to flow cytometric analysis as described above. Absolute numbers (AN) of TS(IYNVGQVSI)/H2Kd tetramer positive cells present in expanded cultures were determined as follows: total cell yield after expansion as determined by trypan blue cell count × (# of TS(IYNVGQVSI)/H2Kd tetramer positive events ÷ number of live events collected). These ANs were used to calculate the antigen-specific AN index (AN Index = absolute number of TS(IYNVGQVSI)/H2Kd tetramer positive cells after A20-TS expansion ÷ absolute number of TS(IYNVGQVSI)/H2Kd-tetramer positive cells present after NC A20 expansion).

Statistical analyses

Results

Construction and functional testing of the IL-15 plasmid DNA

The murine IL-15 gene containing the long signal peptide (LSP) was cloned into the mammalian expression plasmid pVAX as described in Materials and Methods. We inserted an optimized signal sequence for IL-15 which includes a stronger Kozak sequence, removal of upstream inhibitory AUG sequences, and changed the LSP to the Homo Sapiens immunoglobulin secretory leader signal as previously described [16]–[18]. The complete amino acid sequences and protein schematics of the IL-15 genes are shown in Figs. 1A and 1B. Plasmid DNA encoding murine IL-15 sequences with or without the upstream optimization sequence (pIL-15LSP and pIL-15opt, respectively) were transiently transfected into COS-7 cells, and after 48 hours cell lysates were prepared and analyzed for IL-15 expression using IL-15-specific western blots and IL-15-sensitive cells (CTLL-2 bioassays). COS-7 cells transfected with the optimized IL-15 plasmid but not with the pVAX alone expressed IL-15 as shown by western blot (Fig. 1C). The functional activity of the COS-7 synthesized IL-15 is shown in Fig. 1D. These results demonstrate that the cloned optimized murine IL-15 gene can be highly expressed and has potent functional activity in mammalian cells (pIL-15opt referred to as pIL-15 hereafter).

Shown in panels A and B are amino acid alignments and mature protein schematics of the murine IL-15 genes containing the long signal peptide (LSP) and optimized signal sequence (opt). These proteins were cloned into the eukaryotic expression plasmid pVAX and resulting plasmids transfected into COS-7 cells. After 48 hours, lysates from pVAX and pIL-15opt transfected COS-7 cells were prepared and analyzed by western blot along with recombinant murine IL-15 (rmIL-15, panel C). COS-7 cells transfected with pIL-15opt resulted in high level expression of murine IL-15. IL-2/IL-15 dependent cells (CTLL-2) were cultured in 96 well plates with transfected COS-7 lysates in order to determine the functionality of the cloned IL-15 constructs. Purified recombinant IL-15 was utilized as a positive control and used to prepare a standard curve. After 24 hours of culture, 3H-thymidine was added and one day later plates were harvested, scintillated, and radioactive incorporation measured. COS-7 cells transfected with pIL-15opt express functionally active IL-15 as determined using this CTLL-2 bioassay (D).

After demonstrating that co-administration of pIL-15 with pTS greatly enhances long-term protection against T. cruzi challenge, we further dissected T cell responses induced by vaccination with pTS alone compared with pTS + pIL-15. In order to study T cell compartment cytokine production potentials, we stimulated spleen cells from vaccinated mice (6 months after the 3rd and final vaccination) with PMA + ionomycin, then analyzed the overall cytokine production in T cells using intracellular flow cytometry. As seen in Table 1, in general, pTS vaccination alone did not significantly alter CD4+ or CD8+ T cell profiles in comparison with control pIL-15 vaccinated mice in terms of IL-2, IFN-γ, or TNF-α expression. However, vaccination with pTS + pIL-15 resulted in significantly increased frequencies of all cytokine parameters analyzed in both CD4+ and CD8+ T cell compartments (IL-2, IFN-γ, and TNF-α, P<0.05 by Mann Whitney comparing pTS+pIL-15 to pTS alone and pIL-15 alone). In addition, we detected significantly increased frequencies of multi-parameter CD4+ and CD8+ T cells which expressed IL-2 + IFN-γ as well as TNF-α + IFN-γ (P<0.05 by Mann Whitney comparing pTS+pIL-15 to pTS alone and pIL-15 alone).

As total TS-specific IFN-γ T cell responses were found to be greatly increased after addition of pIL-15 to the pTS vaccination regimen 6 months post-vaccination, and these increased responses were associated with increased protection, we next sought to investigate whether pTS-induced CD8+ T cells alone could transfer protection against lethal T. cruzi challenge. The frequencies of CD8+ T cells present in mice vaccinated with pTS DNA with or without pIL-15 have not been sufficient to allow practical adoptive transfer experiments to be done. We have recently found that a strategy of boosting pTS responses with TS-expressing adenovirus vaccinations results in up to 10-20 fold more TS-specific CD8+ T cells. Therefore, we vaccinated BALB/c mice with 2 doses of pTS DNA followed 1 month later with 2 doses of Adeno-TS to obtain sufficient numbers of TS-specific CD8+ T cells for adoptive transfer experiments. As shown in Fig. 6A, mice vaccinated with pTS followed by Adeno-TS developed massive CD8+ TS-specific IFN-γ responses (>22,000 IFN-γ spot forming cells per million CD8+ T cells). We adoptively transferred 2.6×107 CD8+ T cells obtained from negative control or TS prime/boost vaccinated mice into naïve BALB/c recipient mice followed by challenge of these mice the following day with a lethal dose of T. cruzi (5,000 BFT s.c.). As seen in Fig. 6B, mice adoptively transferred with negative control T cells succumbed rapidly to T. cruzi infection (100% mortality within 3 weeks). However, all of the mice which received TS-specific CD8+ T cell transfers survived lethal T. cruzi challenge (P < 0.05, Fisher's exact test). Thus, vaccine-induced TS-specific CD8+ T cells alone can protect against lethal systemic T. cruzi challenge, and the increases in TS-specific CD8+ T cell responses seen in mice vaccinated with pTS + pIL-15 could be sufficient to explain the mechanism for prolonged enhanced protection.

Discussion

The addition of IL-15 adjuvants to vaccine regimens has previously been shown to enhance protective immunity against a variety of pathogens, including influenza, Toxoplasma gondii, and Herpes simplex virus (HSV) [22]–[24]. We have demonstrated here that the addition of a plasmid DNA expressing murine IL-15 (pIL-15) to a plasmid DNA vaccine encoding the T. cruzi trans-sialidase (pTS) leads to significant enhancement of long-term protective immunity against this complex intracellular protozoan pathogen. The combination of pIL-15 and pTS induced significantly higher levels CD4+ Th1 cells and TS-specific IFN-γ producing CD8+ T cells which displayed enhanced proliferative capacity after antigenic restimulation compared with the levels of these responses induced by vaccination with pTS alone. More importantly, these TS-specific immune responses enhanced by IL-15 co-administration led to significant increases in protective immunity 6 months post-vaccination.

IL-15 is a member of a family of common γ chain (γc)-dependent cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, which require expression of the γc receptor component for functional cytokine signaling. The IL-15 and IL-2 receptor complexes also share CD122 (IL-2Rβ/IL-15Rβ) resulting in very similar in vitro effects of these 2 cytokines on the growth of NK cells and different subsets of T cells. However, IL-15 signaling is unique among the γc cytokines involving a plasma membrane/endosomal recycling of the complex between IL-15 and its unique IL-15Rα receptor component which results in the prolonged capacity for IL-15 to signal in trans to cells expressing the γc/IL-15Rβ complex on their surface [25]. In contrast, efficient signaling by soluble IL-2 requires binding to the high affinity IL-2R complex (including CD25/γc/IL-2Rβ) followed by receptor/cytokine internalization and degradation of the IL-2 ligand. Hence, IL-15 signaling is prolonged and dependent on cell-to-cell contact while IL-2 signaling is more transient and requires active IL-2 secretion by activated cells. These biological differences and the distinct expression profiles of CD25 and IL-15Rα likely explain the opposing effects of IL-15 and IL-2 identified in vivo [26]. Deficiency of IL-15 leads to a profound depletion of NK cells, NKT cells, intestinal intraepithelial lymphocytes and CD8+ memory T cells, whereas deficiency of IL-2 leads to lymphoproliferative and autoimmune disease. In addition, IL-15 has overlapping functions with IL-7 important for the survival, maintenance and homeostatic proliferation of both CD4+ and CD8+ memory T cells [26].

The enhancing effects of IL-15 on memory T cell survival and expansion have been viewed as reasons to investigate the potential vaccine adjuvant effects of this cytokine. Our results showing that significantly increased numbers of TS-specific CD8+ T cells persist 6 months after vaccination with pTS plus pIL-15 compared with vaccination with pTS alone (Figure 5) are consistent with the known T cell survival and homeostatic proliferative effects promoted by IL-15. These effects could be due to survival effects triggered in the presence of increased IL-15 during the initial TS-specific T cell activation or maintenance effects provided by prolonged production of IL-15. We were unable to detect increased IL-15 in the serum of mice 6 months post-vaccination with pTS plus pIL-15 (data not shown), but these results do not rule out the possibility that persistence and/or prolonged recycling of surface bound IL-15 occurs on professional antigen presenting cells (e.g. monocytes and/or dendritic cells) expressing the IL-15Rα chain as reviewed above. This would be hard to disprove since IL-15 trans signaling has been shown to provide a highly potent mechanism of signaling through both IL-15Rα/IL-15Rβ/γc trimeric and IL-15Rβ/γc dimeric receptor complexes, active with subpicomolar concentrations of IL-15 [25]. Longer-term and more detailed studies are required to evaluate the maximal duration of the enhancing effects of our pIL-15 adjuvant, and to determine the associated period of time of enhanced IL-15 signaling.

As mentioned above, enhanced expansion and survival of CD4+ memory T cells could provide greater helper effects for TS-specific B cell responses. Alternatively, it is possible that IL-15 could directly enhance antibody responses by signaling through the IL-15Rα known to be expressed on activated B cells [28]. Furthermore, IL-15 has been demonstrated to stimulate proliferation and antibody secretion in B cells facilitated by the inhibition of FAS-mediated apoptosis [29]. However, it seems unlikely that the TS-specific B cell/antibody responses were responsible for the pIL-15-enhanced long-term protection against parasite challenge. We observed potent TS-specific antibody responses 6 months after vaccination in mice given pTS alone, but these mice were not protected against lethal T. cruzi challenge. Furthermore, we have shown previously that vaccination of B cell knockout mice with pTS alone confers protection against T. cruzi challenge [3]. Thus, antibody responses do not seem to be the likely mediator of enhanced protective immunity induced by IL-15 and TS encoding plasmid vaccines.

We were impressed by the enormous differences in PMA/ionomycin-induced cytokine responses in both CD4+ and CD8+ T cells primed by the combination of pTS plus pIL-15 compared with priming by either pTS or pIL-15 alone (Table 1). We detected 4–10 fold increased frequencies of T cells capable of producing IFN-γ, TNF-α, and/or IL-2 after pTS plus pIL-15 vaccination compared with either pTS or pIL-15 given alone. In fact, over 50% of CD4+ T cells from pTS plus pIL-15 vaccinated mice produced TNF-α after PMA/ionomycin stimulation (compared with <12% of CD4+ T cells from mice vaccinated with pTS or pIL-15 alone). We must emphasize that this remarkable enhancement of polyclonal T cell responses required immunization with both pTS and pIL-15 and was not seen in mice vaccinated only with pIL-15. Total percentages and numbers of CD44+ memory CD4+ and CD8+ T cells were nearly identical in pIL-15 alone, pTS alone and pIL-15 plus pTS vaccinated mice (data not shown), indicating that enhanced cytokine responsiveness in pIL-15 plus pTS vaccinated mice was not due simply to an IL-15-induced expansion of pre-existing polyclonal memory T cells. Overall, these results suggest that in the context of antigen-specific stimulation of T cell responses in vivo, increased levels of IL-15 could result in a marked shift of polyclonal T cells into a more activated state with enhanced responsiveness to inflammatory stimuli. Theoretically, this effect could result in a more broadly reactive epitope spreading of immune responses important for optimal vaccine-induced protection against pathogens with high potential for epitope mutational escape. On the other hand, this increased polyclonal T cell responsiveness could lead to increased risks of autoimmunity or immune-mediated inflammatory disease as adverse effects associated with IL-15 adjuvanted vaccinations or immunotherapies. Future studies will need to explore these possibilities in detail.

In summary, our results demonstrate that a plasmid expressing IL-15 can have significant adjuvant effects for induction of enhanced protective T. cruzi immunity and suggest important mechanistic explanations for these effects that should be further analyzed in future research.